During the course of the preceding grant with the same title we have completed sequences accounting for about 250 nucleotides in the structural genes and unambiguously assigned over 100 nucleotides to the untranslated regions. The entire catalogue of fragments resulting from G cleavage of in vitro synthesized RNA has been sequenced, and a substantial start has been made on the C and U cleavage catalogues. Most important, our success in inserting hemoglobin mRNA sequences into bacterial plasmids makes possible new approaches which should enable us to finish the sequences of the globin mRNAs quickly. We will continue our work to complete the globin mRNA sequences, as mentioned above, but there will be a major emphasis on investigation of the DNA sequences extending in both directions from the globin structural genes. In order to accomplish this, we propose novel methods for the rapid purification of specific single-copy gene sequences prior to cloning. Cloning these sequences will enable us to ask detailed questions about control signals, HnRNA precursors, and transcriptional start and stop signals in vivo. Our experience with nucleotide sequence analysis will enable us to make detailed studies of such signals wherever they are localized. The National Cancer Plan has pointed out that there are many areas where our ignorance of normal cellular processes is so profound that we cannot yet even formulate the most penetrating questions about cancer. The work we propose may shed light on some of the most important processes. But especially important, the methods we propose to develop will be useful in the direct study of integrated tumor virus sequences. The gene isolation techniques we propose could be immediately useful for such purposes, but biohazard considerations presently preclude the application of the cloning techniques to oncogenes. As explained in Appendix 1, however, new developments in biocontainment techniques promise levels of sophistication far beyond present EK-2 specifications. This may permit the entire range of techniques which will develop for the globin genes to be applied to oncogenes.